Method and apparatus for processing nucleic acids using a small temperature-changing zone

ABSTRACT

PCT No. PCT/EP95/00975 Sec. 371 Date Sep. 19, 1996 Sec. 102(e) Date Sep. 19, 1996 PCT Filed Mar. 16, 1995 PCT Pub. No. WO95/25592 PCT Pub. Date Sep. 28, 1995Method and device for processing nucleic acids in a reaction mixture on a surface that can be temperature-controlled and its immediate vicinity wherein the main space of the reaction mixture remains essentially isothermal. The method has the advantage of a very short processing time.

The invention concerns methods for processing nucleic acids by means ofa temperature regulation element as well as devices and instruments forcarrying out these methods.

In analytics and especially in medical diagnostics more and more methodsare beginning to be established which are based on nucleic acid testsand syntheses. Nucleic acids are for example very well suited as a veryspecific detection agent for organisms for the diagnosis of diseases.During these detection methods various processing steps such asdenaturation, hybridization, syntheses and immobilization of nucleicacids and enzymatic treatment thereof are common. For a long time aproblem of such methods was the small amount of nucleic acids in thesamples.

A solution for this problem, which has made it possible to detectnumerous analytes, was the amplification of nucleic acids. Such a methodis described in EP-A-0 200 362 i.e. the polymerase chain reaction (PCR).In this method many copies of the original nucleic acid are produced byrepeated extension of primers in a reaction solution. These can forexample be detected by hybridization with a labelled nucleic acid probeaccording to EP-A-0 201 184. The reaction solutions used for this areheated to particular temperatures and cooled again at intervals in orderto separate double strands and for the extension reaction. Since thevolumes are relatively large, the times required for temperatureregulation result in a relatively long period to carry out the entireamplification process.

It has been attempted to remedy this by so-called capillary PCR. In thismethod the reaction mixture is present in glass capillaries of a smalldiameter. As a result the time required to carry out an amplificationsequence can be reduced to ca. 30 min. A problem with capillary PCR isthe delicacy of the glass of the capillary which also has to be heatedand the cumbersome sample application.

Recently more and more amplification processes are also being describedin which the amplification reactions can be carried out in a closedsystem i.e. without supplying reagents during the cycles. Thus in asystem is described WO 92/07089 in which the amplification mixture ispassed as long as necessary through a circulation system in which thereaction mixture is repeatedly heated and cooled. The period in whichthe mixture is retained in the individual zones of the system can beinfluenced by selecting the diameter. An amplification process isdescribed in EP 0511712A1 in which the amplification mixture is broughtcyclically to quite specific temperatures in order to achieve relativelyshort cycle times. In this case also the sample handling is made moredifficult.

The object of the present invention was among others to improve theexisting nucleic acid processing methods and in particular to provideprocesses in which the amplification can be completed in a particularlyshort time.

The invention therefore concerns a method for processing and inparticular amplifying nucleic acids in a reaction mixture characterizedin that the temperature of a surface adjoining the reaction mixture andits immediate surroundings is regulated but the main space of thereaction mixture remains essentially isothermal.

The invention also concerns a device for processing and amplifyingnucleic acids with a temperature regulation element as well as aninstrument which contains this device.

Nucleic acids within the sense of the invention are all types of nucleicacids, modified or unmodified. Unmodified nucleic acids are for examplethe naturally occurring nucleic acids. Modified nucleic acids can beformed by substituting groups of the natural nucleic acids by otherchemical residues. Examples are nucleic acid phosphonates orphosphothioates and nucleic acids modified on their sugar residues orbases by chemical groups which may also be detectable.

The processing of nucleic acids according to the present inventionpreferably includes at least one reaction step which proceeds at anincreased temperature that is at least different from the ambienttemperature. Such reaction steps are for example the thermaldenaturation of partially or completely double-stranded nucleic acids.In this process the nucleic acids are heated to temperatures above therelevant melting point in order to produce single strands or to meltsecondary structures. A further processing step of nucleic acids is thehybridization of the single-stranded regions of nucleic acids which arecomplementary to one another to form a nucleic acid double strand(hybrid). This takes place at temperatures below the melting point ofthe hybrid. Therefore in-order achieve a hybridization it is oftennecessary to cool the reaction mixture. Especially in amplification orsequencing methods a further processing step is carried out namely theextension of a primer hybridizing to a so-called template nucleic acidwith the aid of further mononucleotides or oligonucleotides. In t hiscase the temperature is preferably adjusted to that at which the enzymeused has its activity optimum or at which competing reactions arereduced. This temperature may also be identical to the hybridizationtemperature (2-step PCR).

A special case for processing nucleic acids is the amplification ofnucleic acids. In this connection practically all amplification methodse.g. target sequence-dependent amplifications (in particularnon-isothermal amplifications such as the polymerase chain reaction,ligase chain reaction or similar ones) can be carried out according tothe invention. Also during these amplification methods at least one ofthe aforementioned temperature sensitive processing steps takes place.

A key feature of the present invention is the fact that the change oftemperature does not take place in the entire reaction mixture but onlyin a very small part of the reaction mixture. As a result the heatingand cooling of this relatively small zone can occur very rapidly thusconsiderably accelerating the processing of nucleic acids. Therefore inorder to carry out the process according to the invention the reactionmixture is contacted with a surface whose temperature can be regulated.By changing the temperature of the surface the immediate surroundings ofthis surface are heated, equalized or cooled depending on the processingstep and temperature of the reaction mixture. One can differentiatebetween several cases.

If the temperature of the reaction mixture is higher than the meltingtemperature of the nucleic acids to be processed, the surface can becooled in order to achieve a hybridization of the nucleic acids in theadjoining vicinity. If the temperature of the reaction mixture is lowerthan the melting temperature and the temperature which is required toextend a primer on the nucleic acid, the surface and thus the immediatevicinity can be firstly heated to a temperature at which an extension ofthe primer can take place. Subsequently the temperature can be increasedto above the melting point of the nucleic acids which enables adenaturation and strand separation of the double-stranded nucleic acidswhich have been formed. This can be followed by cooling to a temperatureat which the single strands can hybridize with new primers. This cyclecan be repeated several times.

If the temperature for the reaction mixture lies between the optimaltemperature for extension and the melting temperature, the temperatureat the surface is firstly increased in order to separate double strands.Subsequently the temperature is lowered to a temperature at which thesingle strands hybridize with primers. Subsequently the temperature isadjusted to an optimum for the extension and if desired the cycle isrepeated.

The desired temperature can be maintained over a predetermined periode.g. until the desired reactions have taken place at the surface. Forthis purpose the temperature of the surface can also be kept constant byknown control measures and connected regulation measures (reheating,recooling). The time periods depend, as is usual for the knownprocesses, on the length of the nucleic acids to be processed and theirhomology as well as on the special hybridization conditions. However, aperson skilled in the art can determine the optimum periods by simpleexperiments. The time periods are between fractions of seconds and a fewseconds depending on the processing step.

In the above mentioned cases the main space of the reaction mixtureremains essentially isothermal whereas the reaction mixture located inthe immediate vicinity of the surface adjusts to the set temperatures ofthe surface.

In order to regulate the temperature and to set specific temperatures atthe surface and its immediate vicinity it is recommended that a surfaceof a device is selected as the surface that is composed of a heatingelement and a cooling element. The heating element preferably has arelatively large surface with a comparatively small heat capacity. Metalfoils have for example proven to be suitable which can be heated in asuitable manner e.g. by electrical current. Good heat conductingmaterials such as gold are preferred as materials for the metal foil.

The cooling element should have a relatively high heat capacity. Solid,liquid or gaseous substances come into consideration as the coolingmedium. Liquid cooling media such as water are preferred. A good heatconductivity is advantageous.

The arrangement of the heating element and cooling element can bechanged according to the temperature of the reaction medium depending onwhether it is intended to use the device more for heating or for coolingthe surface and its immediate vicinity. However, in general the heatingelement is located in the immediate vicinity of the surface. The surfaceof the heating element can also directly adjoin the reaction mixture.However, it is also possible to separate the heating element from thereaction mixture by a thin heat conducting layer.

The cooling element can in principle be positioned at any position whichallows a cooling of the surface and the immediate vicinity. However, ithas proven to be advantageous to position the cooling element on theopposite side of the reaction mixture to the heating element. If theheating element has a low heat capacity, the fact that the heatingelement must also be cooled is not an important disadvantage.

In the method according to the invention the heating element is used toheat until the surface and its immediate vicinity have been heated tothe desired temperature. In this process a pronounced temperaturegradient will form near the surface whereas the remaining part of thereaction mixture remains isothermal. This applies in particular if thereis no significant exchange of liquid during the heating process but evenif there is an exchange of liquid. The heating process can be completedwithin fractions of a second and in favourable cases even ismilliseconds. The same applies to the cooling. The reaction space whichis heated to the desired temperature can be very small, it is preferablyless than 0.2 mm particularly preferably less than 0.5 μm deep. The areaof the heating element facing the reaction mixture influences the depthof the temperature gradient and its rate of formation. A preferred sizeof the surface may result from the desired application of the process.Usually the surface is <2 cm², particularly preferably between 0.2 cm²and 0.2 mm². The surface can be smooth or also rough. If the surface isalso used at the same time as a carrier for agents for processingnucleic acids it is advantageous to enlarge this surface by surfacestructures.

The volume of the reaction mixture is of no major practical importancefor the invention. It may be a drop with a volume of 20 μl but also avolume of any size. An advantage of the process according to theinvention is that the device containing the heating and cooling elementscan for example also be inserted into a large vessel containing thereaction mixture in which case the key reactions only take place in avery small boundary region of the surface whereas the remaining reactionspace exerts almost no influence on the reaction. On the other hand thesamples that are available and amounts of reagent represent a practicallimit to the size of the reaction mixture. The reaction volumes willtherefore usually lie in a range between 1 ml and 30 μl preferablybetween 100 μl and 50 μl but it is also for example possible to use verymuch smaller volumes by applying the reaction mixture onto thetemperature-regulating surface.

In order to process nucleic acids these are applied to thetemperature-controllable surface or its immediate vicinity. This can beachieved in various ways for example mechanically or by diffusion. Thenucleic acids are preferably bound to the surface. This binding can inturn be of various types. A chemical binding is preferred either viaadsorption or biospecific interactions. In the case of binding byadsorption, the surface can be covered with a nucleic acid bindingreagent. Biospecific interactions can be interactions between nucleicacids and the nucleic acids to be processed (hybridization,complementary part of these nucleic acids) but also interactions betweenantigens or haptens and antibodies directed against them andreceptor-ligand interactions. A preferred type of binding of the nucleicacids to be processed to the surface is via oligonucleotides which arebound covalently to the surface and which are complementary to at leastpart of the nucleic acid to be processed. However, theseoligonucleotides can also be bound to the surface by means ofbiospecific interactions e.g. biotin-streptavidin.

According to the present invention the binding of the nucleic acids tobe processed is preferably reversible. The nucleic acids can be releasedagain after a period depending on the process which is to be carried outby heating the surface and its immediate vicinity. Any desired step canbe carried out between the binding and release of the nucleic acid suchas chemical reactions and also a separation of the bound nucleic acidsfrom the original reaction mixture and transfer into a new reactionmixture.

In the case of an amplification reaction of nucleic acids theoligonucleotides bound to the surface can be used as primers forelongation or extension using the nucleic acid to be processed as atemplate. As a result a bound extended primer forms on the nucleic acidto be processed. The nucleic acid used as a template can be detachedfrom the extension product by increasing the temperature and in the nexttemperature cycle it can act as a template for a new immobilized primerwhich has not yet been elongated. In this manner it is possible toextend a large amount of primers immobilized on the surface and thusproduce copies of parts of the nucleic acid to be processed. Theextended (elongated) primers attached to the surface in turn serve tomultiply the molecule serving as a template by means of complementaryprimers. The reagents required to carry out the reactions have either tobe kept on hand in the total reaction mixture or they must be added asrequired. Therefore for an amplification reaction according to theprinciple of a polymerase reaction (EP-B-0 201 184) thedeoxyribonucleotides, a DNA polymerase and a further primer and suitablebuffer reagents must be kept in the reaction mixture. In order toprepare other nucleic acid types (e.g. RNA) other reagents e.g.ribonucleotides or RNA-dependent polymerases are provided. This step ofthe process can take the working temperature of the processing enzymeinto particular consideration.

The nucleic acids to be processed can, however, also be bound to thesurface by physical methods e.g. by means of a magnet. For this thenucleic acids must, however, be bound to a magnetizable particle. Thebinding of magnetic particles coated with nucleic acid can be carriedout in a reversible manner by locating or inducing a magnet behind thesurface. By applying an alternating field it is possible to bind themagnetic particles to the surface or remove them therefrom.

Special embodiments which have some advantageous effects are alsoconceivable. Thus the efficiency can be increased by increasing thediffusion of the nucleic acids in the reaction mixture by convection. Itmay also be advantageous to use higher concentrations of the reactantscompared to the reaction mixtures usually used. In addition the nucleicacid to be processed can be concentrated at the surface at the start ofthe reaction by prehybridization.

In the case of an amplification or multiplication of nucleic acids itmay be that the number of cycles to be carried out e.g. in a PCR has tobe increased in order to produce sufficient amplification product.However, due to the very short cycle time according to the process ofthe invention this is not a disadvantage.

If the reaction mixture is kept at a relatively low temperature thismeans that less demands have to be made on the heat stability of thereagents than when the entire reaction mixture is heated several times.Therefore a thermostable polymerase is not absolutely necessary for anamplification reaction and nevertheless it is not necessary to pipettenew enzyme into the reaction mixture in each amplification cycle.

Any desired further steps can follow the processing of nucleic acidsaccording to the invention. Thus the nucleic acids can be examinedeither in a bound state on the surface or in a released state orprocessed with further reagents.

Thus with the aid of the described amplification process according tothe invention it is possible in a simple manner to develop a process fordetecting nucleic acids in a sample. For this the surface to which theoligonucleotides acting as a primer are bound is contacted with thesample liquid. Afterwards the temperature of the surface and itsimmediate vicinity is brought to a temperature which lies above themelting point of the double-stranded nucleic acid contained in thesample. After cooling the surface and its immediate vicinity the nucleicacid to be detected is hybridized with the oligonucleotide. By means ofthe cycle process as described above numerous copies are produced withthe aid of the nucleic acid to be detected which can remain bound to thesurface at the end of the amplification reaction. The amount of nucleicacids on the surface can be determined by hybridization with labellednucleic acid probes and detecting them with the aid of the label. In thecase of methods which allow a direct detection of nucleic acid hybridswithout a label or of extended single strands (primers) (e.g. accordingto the principle of surface plasmon resonance) a direct detection isalso possible. If labelled primers or mononucleotides are used for theamplification reaction hybridization with a detectable labelled nucleicacid probe is omitted. An approach is also conceivable in which a highertemperature is maintained in the mixture and the surface adopts thelower temperatures necessary for the further steps.

Since the application of the process according to the invention toamplify nucleic acids proceeds practically on a surface, we shallintroduce the name 2D amplification for this amplification reaction.

The invention also concerns a device which can be used in theaforementioned processing method. In particular the invention concerns adevice for processing nucleic acids by means of a temperature regulationelement, this element being suitable for regulating the temperature ofthe surface and its immediately adjoining vicinity and wherein agentscan be bound to this for processing the nucleic acids. These agents canbe oligonucleotides and serve for example as primers. This devicepreferably contains a cooling element as well as a heating element inwhich the arrangement is preferably as in the processing methoddescribed above. This device is preferably very small. It can forexample have a thickness of <5 mm and an area of <10 cm². The elementslocated therein such as the cooling element or the heating element aresimple components. Therefore this device is excellently suitable forsingle use (disposable) which can reduce the risk of contamination thatis inherent to re-usable devices. If the device is to be suitable formultiple use, it is also possible to separate the heating element by avery thin component from the space which contains the reaction mixtureand to make this component separable from the device. A new componentcan then be inserted for a further processing step.

The invention also concerns an instrument for processing nucleic acidswhich contains a control element for a time-dependent temperatureregulation as well as a device according to the invention. The controlelement of a so-called thermocycler according to EP-A-0 236 069 can beused as the control element; however, a control according to theprinciple of ink-jet printers is preferred since they have a highercontrol rate. The control element must be able to heat the heatingelement at predetermined intervals until the vicinity of the surface hasan adequate temperature. It must also be able to provide a cooling ofthe surface by means of a cooling element at predetermined intervals.For this a cooling liquid can for example be passed through the device.Provided the heat capacity of the heating element is small but theheating efficiency is relatively high, a continuous cooling is alsopossible. Various forms can be used as the surface in an ohmicallytemperature controlled version of this instrument which can be thermallyheated depending on the voltage and the duration of the current. In amodified version the surface to be temperature controlled is located ona preferably black support, preferably a plastic foil, which is heatedby a laser ray, preferably an infrared laser, on the side distal to thesurface to be temperature controlled. The surface to be temperaturecontrolled as well as the heat-absorbent and heat-conducting materialcan in this case be mounted on a support surface preferably glass thatis permeable to infrared.

The device according to the invention can have various embodiments. Forexample it can be designed to be immersed in a vessel. However, it canalso be designed such that the liquid containing the nucleic acid to beprocessed is dripped onto the surface or is dispensed into a vesselformed by the surface. This reaction space can also be closed afterfilling it with the liquid so that contamination problems can bereduced.

One embodiment of the invention is a process for amplifying nucleicacids. This is as in example 3, for example an amplification reactiondenoted polymerase chain reaction with subsequent detection.

A further embodiment of the process according to the invention is aprocess for concentrating nucleic acids e.g. by hybridization. Whenprimers with an adequate specificity are adsorbed covalently onto thesurface, it is possible to preset a hybridization temperature when thereaction solution to be analysed has a known composition (ioniccomposition, concentration of reagents) such that the nucleic acid to beanalysed binds specifically to the primers bound covalently to thesurface for hybridization. This process can be used

a) to exactly analyse the actual hybridization temperature

b) to achieve an exact concentration of the nucleic acid to be analysedin the reaction mixture

c) to test for the presence of cross-hybridizing sequences.

This short list is not definitive.

This process can also be used to transfer nucleic acids from onesolution into another. Then a hybridization takes place in the firstvessel (lower temperature) and a denaturation in the second vessel(higher temperature).

A further embodiment of the present invention is a process forsequencing nucleic acids. One method of applying the invention inrelation to sequence analyses is the so-called minisequence analysis. Inthis method the exact sequence of the nucleic acid nucleotide adjoiningthe primer can be determined by adding exclusively dideoxynucleotidesand no deoxynucleotides for sequencing to the solution which is providedfor sequencing. The four possible dideoxynucleotides ddATP, ddCTP, ddGTPand ddTTP are labelled with different fluorescent labels. In each casethe incorporation of the next respective nucleotide thus leads to atermination of the sequence reaction and, after purification of theextended primer located at the surface, the sequence of the nucleotidelocated on the primer can be determined by analysis of the specificfluorescence.

A special case for the possible application of the present invention isa process for sequencing nucleic acids which have previously beenamplified in a PCR reaction. After the amplification the amplifiedproduct is present covalently bound via the primer to the reactivesurface in double-stranded form. Denaturation of this amplificate by abrief passage through a high temperature phase will make itsingle-stranded, will allow it to be purified by transfer to a washingsolution and can afterwards be used again for a subsequent sequencing bytransfer this time into a sequencing reaction. This sequencing isparticularly efficient since now only single-stranded template materialis present to which a sequencing pair binds particularly efficiently. Asa result of the sequencing reaction a double-stranded molecule issubsequently again present whose labelled (sequenced) half is nowavailable for analysis by gel chromatography. By using different labels(with dideoxynucleotides labelled with different fluorescent markers)all four reactions necessary for a sequence analysis can be carried outsimultaneously; with the conventional method this analysis has to berepeated four times.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is elucidated in more detail by the followingfigures:

FIG. 1 shows the construction diagram of a device according to theinvention (1) which dips into a reaction vessel (3) filled with thereaction mixture (2). The device contains a cooling element (4) and aheating element (5) heated by means of a current. Oligonucleotides (6)are covalently bound to the surface of the heating element. The coolingelement as well as the heating element can be regulated via connections(7; 8) to control units.

An example of a temperature curve for an amplification reaction of thePCR type is shown in FIG. 2. In a first phase (A) the nucleic acids aredenatured at a temperature of slightly below 100° C. In a phase (B) thenucleic acids to be amplified are hybridized with the immobilizedoligonucleotides on the solid phase at about 50° C. In a third phase (C)the primers are extended at ca. 70° C. using the nucleic acid as atemplate.

FIGS. 3A-3C shows the reactions occurring in the immediate vicinity ofthe surface (phases A to C). Three isotherms are shown as a schematictime course for phase (A) and only one isotherm is shown for phase (B)and (C) in each case. The nucleic acids are denatured in phase (A) anddiffuse for a certain time as single strands, in phase (B) templates andprimers hybridize and in phase (C) primers are extended along thetemplates.

FIG. 4 shows a possible prototype of a surface temperature controlsuitable for an application according to the invention.

The heating unit which contains the replaceable heated gold surfaces aswell as the physical requirements for cooling or heating (connection forcooling agent, electrical connection) is attached via plug connections(7; 8) to the actual measuring unit (1) to which holders for reactionvessels (11) are also attached in which the template molecules, buffercomponents and primers in solution and the polymerase required forcarrying out the reaction are present.

The measuring sensors (9) in this case in the form of a test stripcontain as an example gold surfaces (5) in FIG. 4 which do not have tobe coated with the same primers (6).

FIG. 5 shows enlargements of the measuring units with replaceable teststrips from FIG. 4 in which case the measuring unit (1, composed of 9and 10) is composed of an electronic connection and cooling system(upper, grey part of the figure) and the test strip (=measuring casing).The lower part of the figure shows a cross-section through thismeasuring unit which shows the cooling circulation (4) used for thecooling with a stirring mechanism (12) and cooling liquid (13) as wellas the reaction surfaces (5, 6) with an electronic connection. The righthand part of the lower figure shows a further enlargement of the cathodeconnection of the gold membrane (5) with a supporting and separatingfilter (14) as well as an adhesion layer for the oligonucleotides (6).

FIG. 6 is composed of a diagram of the temperature gradients formed inthe immediate vicinity of the adhesion layer containing oligonucleotidesand the temperature profile which is to be expected, in this case asexemplified by a three-step PCR in which temperatures of 96° C. for 0.1seconds, 54° C. for 0.5 seconds and 72° C. again for 0.5 seconds areshown.

The following examples are intended to further elucidate the inventionin more detail:

EXAMPLE 1

Manufacture of a Surface that can be Cyclically Temperature-Controlled(Variant a)

An exemplary gold surface that can be cyclically temperature-controlledcan be obtained by milling out two parallel oblong holes of ca. 1 mmwidth and 3 mm length at a distance of 3 mm to one another in a thinprinted-circuit board. On the side which is facing the solution andwhich is therefore called the proximal side these oblong holes areconnected to one another by vapour-deposited gold. On the distal sideone of these oblong holes is connected to the anode via thecircuit-board conductors the other is likewise attached to the cathodevia the circuit-board conductors.

The gold layer on the proximal side is applied by applying a mask of thedesired size, in this case 3×3 mm flush, over the two longitudinalsurfaces. The inner sides of the longitudinal surface are galvanicallycoated with copper flush up to the surface. In the followingvapourization process which is carried out conventionally the twoelectrically conductive longitudinal holes are now coated with only afew μm thick gold layer. The coating process is described in thefollowing:

The gold surface is manufactured by vapour-depositing on "Lexan"polycarbonate foils (manufacturer: General Electric, thickness 0.75 mm)with dimensions of 8×8 cm over a metal mask (d=0.5 mm, aluminium) in aLeybold high-vacuum coating apparatus (Univex 450). The surfaces lie atregular distances which enables the vapour-coated plate to be separatedinto several units. The thickness of the gold layer is 300 nm. In afurther step the multi-gold spot plate is coated with "dilute"biotinthiol binding layers to form a hydrophobic SAM layer. Thebiotinthiol compound (HS-C12-DADOO-biotin;N,N'-(12-mercaptododecylyl-biotinylyl)-2,2'-diaminoethylglycol diether;2.94 mg; 5×1⁰⁻⁵ m) and the diluent (12-mercaptoundecanol, 9.2 mg,4.5×10⁻⁴ m) were dissolved in 100 ml ethanol p.a. The freshly coatedpolycarbonate foils were immersed in this solution. After 4 hours theplates were taken out, washed twice with ethanol p.a. and immediatelycoated with streptavidin. For this purpose the gold spots and theSAM-coated foils were immersed for 1 hour in a streptavidin solution(streptavidin concentration: 0.5 mg/ml in 0.05 M potassium phosphatebuffer pH 7.2). The surfaces treated in this way were subsequentlytreated with a washing solution (50 mM potassium phosphate buffer pH7.2, 2% sucrose, 0.9% NaCl, 0.3% bovine serum albumin fraction II) andafterwards dried for 20 h (25° C. and 40% humidity).

The very small thickness of the gold layer results in a very smallconducting gold cross-section (3 mm×3 μm over a length of 3 mm). Thisgold layer therefore represents a relatively high resistance compared tothe circuit-board conductors.

The conductors are now connected to a current supply which enables thecomputer-aided control of the ohmically induced temperature of the goldsurface.

Manufacture of a Surface that can be Cyclically Temperature-Controlled(Variant B)

In one variant of the manufacture of the surface that can be cyclicallytemperature-controlled a gold layer is vapour-deposited on the coveringlayer which isolates the measuring meander made of platinum, acommercially available temperature sensor made of platinum (PT 100). Ifthis measuring sensor is now connected to a regulator-controlled voltagesource then this sensor can also be used as a heat source for thevapour-coated gold layer. For this it is necessary to pass the currentand voltage of the platinum sensor via an analogue divider onto aregulator. The regulated parameter of the regulator is the resistance ofthe heated platinum element which enables an exact temperature controlwhen normalization of the gold surface temperature with the temperatureof the platinum meander has taken place.

EXAMPLE 2

An Example of a Device to Measure the Surface Temperature of a Gold Foil(Variant a)

A gold foil according to example 1, variant a is covered with a maskwhich allows a ca. 1 mm wide gold thread to be vapour-deposited on thegold foil which protrudes beyond this gold surface and can be connectedto a measuring point. A further mask which allows a second thread to bevapour-deposited e.g. of nickel, bismuth or another alloy suitable fortemperature measurement, is also prepared. The metal thread lies on thesame side of the gold surface as the gold thread (200 nm thick) but itspath separates when leaving the gold surface to betemperature-controlled and it leads to a second measuring point as aseparate metal thread (300 nm thick). This arrangement of the measuringprobes made of gold and a further metal or alloy suitable fortemperature measurement enables the thermovoltage (2.2 mV/100°0 Kalvin)to be measured in the area of the gold foil where the two threads of themeasuring probe overlap. The temperature at the cold junction ismeasured with a thermoprecision PT 1000 element (accuracy >1%) andstandardized to 10 V (0 V corresponds to 0°, 10 V corresponds to 100°),only amplified and also standardized to 10 V. The sum of the twotemperatures is formed in a summation amplifier and used toelectronically regulate the respective surface temperature.

EXAMPLE 2b

An Example of a Device for Measuring the Surface Temperature of the GoldSurface From Example 1, Variant b

In order to exactly determine the surface temperature of the gold layerit is possible to press together the surfaces of two PT 100 measuringsensors vapour-coated with gold as described in example 1 variant b insuch a way that half of the gold-coated area of the heating sensorcovers half the area of the measuring sensor and the remaining area ineach case is located in the surrounding medium. If only one sensor isnow used for measuring and the other one for heating this then allowsthe temperature of the gold surfaces to be calculated exactly. Thetemperature at the surface of the sensors exactly corresponds to themean temperature of the heated and unheated sensor which can both beexactly defined by measuring their resistance. In this way using atemperature sensor used for measurement it is possible to calibrate awhole series of temperature-controllable surfaces and prepare them forcarrying out PCR reactions. The temperature gradient between the heatedand unheated surface of two surfaces arranged in this manner is ca. 3°C.

EXAMPLE 3

Procedure for a Two-Step PCR Using a Primer that is Immobilized to aSurface

An example of carrying out a polymerase chain reaction uses abiotin-labelled primer that is immobilized on a surface(5'-GAAGGGAGGAAGGAGGGAGCGGAC-3'SEQ ID NO: 1). The 5' end of this primeris coupled via its biotin group to a superficial streptavidin orgold-streptavidin surface. The molar amount of streptavidin orbiotin/square millimeter is about 0.2 pmol/mm². Nanogram amounts or lessof the following double-stranded or single-stranded template molecule(only one strand is cited5'-GAAGGGAGGAAGGAGGGAGCGGACGTCCACCACCACCCAACCACCCCACCC-3'SEQ ID NO: 2)and an opposite primer at a concentration of between 0.5 μM and 2 μM arepresent in solution. The reaction takes place in commercial PCR buffer(Boehringer Mannheim, instruction enclosure for the enzyme, 8th edition,order no. 1146165) at 1.5 mM Mg²⁺. A commercial Taq-DNA polymerase(Boehringer Mannheim, order no. 1146165) at a concentration of 20 nM isused as the polymerase. The opposite primer(5'-GGGTGGGGTGGTTGGGTGGTGGTG-3'SEQ ID NO: 3) present in solution waslabelled with digoxigenin at its 5' end (Patent EP 0324474).

The PCR reaction was carried out at a constant solution temperature of68° and on a primer-coated surface cyclically varying between 96 and68°. The cycle duration of the higher temperature in our example variedbetween 0.1 seconds, 10 seconds and 1 minute, the lower temperaturebetween 0.5 seconds, 20 seconds and 1 minute.

After completing the cyclic heating the solution was cooled to roomtemperature in the process of which the complementary elongatedbiotin-labelled and elongated digoxigenin-labelled primer hybridize toone another and double-stranded DNA fragments are formed. After washingwith PCR buffer solution the surfaces were reacted with anti-Dig-POD,incubated for 30 minutes, washed again and admixed with ABTS (colourprotocol according to the instruction enclosure item 3 of the BoehringerMannheim Reverse-Transcriptase-Assay, non-radioactive, order no.1468120). The coloured product that forms was determined quantitativelyin an ELISA photometer at a wavelength of 405/490 nanometers. Theabsorbance values obtained prove an amplification of a region of thetemplate measured as the extended opposite primer that hybridizes to theprimer coupled to the solid phase. In typical experiments we obtainedabsorbance values of 0.4 when measuring in an ELISA reader and aftercorrecting the digoxigenin-free blank value. The values in controlexperiments without template were 0.04 after correcting the blank valueand in controls without polymerase or without temperature increase butusing the complete reaction mixture the values were 0.07. Even whenheating the surface for several minutes (5 minutes) the ambienttemperature in the constantly temperature-controlled solution onlyincreased by 7° C. even when a significantly (100-fold) larger surface(3 cm²) was heated.

EXAMPLE 4

Manufacture of a Device According to the Invention

An example of a device according to this invention is composed of threestructural units. These three structural units are

1. the heatable temperature-controllable surface which penetrates intothe liquid reaction medium

2. the components that are necessary for heating and cooling that areconnected to

3. the electronic control.

The surface of the element described in this example is composed of athin gold foil with a thickness of less than half a millimeter which isfirmly bound to a streptavidin layer. The biotin-labelledoligonucleotides required for the further processing steps can likewisebe applied to this dextran layer by means of affinity coupling. Theseoligonucleotides are then coupled covalently via their 5' phosphate endto the surface and thus have a reactive 3' end which is accessible toenzymes.

This gold foil which can be structurally stabilized by a supportingplastic net and which has a reactive surface of about 5×5 mm serves atthe same time as the heating element since it is connected to anelectric power source which leads to a spontaneous heating of this goldfoil when current flows. The cooling element is located distally to thereaction mixture, behind the gold foil. In this case the cooling elementis composed of a channel recessed in plastic which adjoins the gold foilwith an area of 7 mm in width. The channel is 2 mm in depth and runsalong the complete length of that part of the instrument denotedmeasuring sensor which in this case comprises the heating element andcooling element as well as the reactive surface. This channel runs fromone end of the measuring sensor to the other end of the measuring sensorat which the reactive surface is applied and back again by inclusion ofa cross-piece that separates the two channels. In this component denotedcoolant loop a suitable cooling liquid e.g. water is circulated. Theentire unit is cast in hard plastic and can be recycled i.e. the goldfoil as well as the plastic are reusable.

The control of the heating the reactive surface as well as of thecirculation rate and precooling temperature of the coolant is achievedby means of a third electronic component which is attached outside themeasuring sensor. This third unit also comprises the storage vessel aswell as the connectors for the cooling liquid. In addition there is amicropump in this third unit which is responsible for circulating thecooling liquid. The measuring sensor is made ready-to-use by flangingthe coolant pipes onto the measuring unit and connecting the powersource to the reactive surface. The individual temperature ranges aswell as the duration of action influence on this surface can bepreselected via an input unit that is attached to the surface of thecontrol unit and has a digital display.

EXAMPLE 5

Procedure for an Amplification Reaction with the Aid of the DeviceAccording to the Invention

This example of an application of the device according to the inventionis an amplification reaction of a predetermined nucleic acid sequencedenoted polymerase chain reaction. Two primers each with a length of 16bases that code at a distance of 4 nucleotides to one another have beenselected for this nucleic acid sequence. The sequence of one of theprimers is identical to and that of the other one is complementary tothe sequence to be analysed. The primer denoted identical in this caseis coupled to the reactive surface via a biotin label present at the 5'end. The other complementary primer as well as all further reagents thatare usually used in a so-called PCR reaction are added to the reactionmixture. The reaction is started by setting the respective reactiontemperatures that are to predominate in the immediate vicinity of thereactive surface and by immersing the reactive surface or the measuringsensor (see example 4) into the reaction mixture. The temperaturechanges in the reactive surface are programmed such that 50 heating andcooling cycles can be carried out within ca. 1 minute. The surface isfreed of all non-covalently bound reaction partners by subsequentimmersion of the measuring sensor into a solution of distilled water andbriefly heating it. For analysis the surface purified in this way thatnow only contains primer molecules and extended primer molecules isintroduced together with the measuring sensor into an apparatus whichenables a direct determination of the amount of extended product basedon plasmon resonance.

LIST OF REFERENCE SYMBOLS

1. device, measuring unit according to the invention

2. reaction mixture

3. reaction vessel

4. cooling element

5. heating element/surface

6. oligonucleotides

7. connections for the cooling element

8. connections for the heating element

9. replaceable measuring sensor

10. electronic connection and cooling system

11. holder for reaction vessel

12. stirrer

13. coolant

14. supporting and separating filter (separating the coolant)

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: nucleic acid                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - -      GAAGGGAGGA AGGAGGGAGC GGAC     - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: nucleic acid                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - -      GAAGGGAGGA AGGAGGGAGC GGACGTCCAC CACCACCCAA - # CCACCCCACC C             51                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: nucleic acid                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - -      GGGTGGGGTG GTTGGGTGGT GGTG     - #                  - #                    24                                                                    __________________________________________________________________________

I claim:
 1. A method for rapid processing of nucleic acids in a reactionmixture, the method comprisingtemperature-regulating the reactionmixture by changing a temperature of a temperature-changing element toeffect a temperature change in a temperature-changing zone which adjoinsthe temperature-changing element, while maintaining the remainingportion of the reaction mixture at essentially isothermal conditionsthroughout the temperature-regulating step, wherein thetemperature-changing element has a surface which adjoins thetemperature-changing zone, the temperature-changing zone is relativelysmall as compared to the entire reaction mixture and comprises an arealess than 0.2 mm in length from the surface, and reagents capable ofbinding to the nucleic acids to be processed are bound to the surface.2. The method of claim 1, wherein the reagents are oligonucleotides. 3.A method for detecting nucleic acids in a reaction mixture,comprisingproviding a reaction mixture containing nucleic acids to bedetected and reactants for processing the nucleic acids in a processingprocedure; temperature-regulating the reaction mixture by changingtemperatures of a temperature-changing element to effect temperaturechanges in a temperature-changing zone which adjoins thetemperature-changing element, while maintaining the remaining portion ofthe reaction mixture at essentially isothermal conditions throughout thetemperature-regulating step, to amplify any nucleic acids in thetemperature-changing zone, wherein the temperature-changing element hasa surface which adjoins the temperature-changing zone, thetemperature-changing zone is relatively small as compared to the entirereaction mixture and comprises an area less than 0.2 mm in length fromthe surface, and reagents capable of binding to the nucleic acids to beprocessed are bound to the surface; and detecting the amplified nucleicacids.
 4. The method of claim 1, wherein at least one reaction stepinvolved in the processing of the nucleic acids proceeds at an increasedtemperature in the temperature-changing zone.
 5. The method of claim 1,wherein the temperature change in the temperature-changing zone is atleast partially accomplished by convection or by stirring the reactionmixture.
 6. The method of claim 1, wherein the reaction mixture furthercomprises further reagents capable of binding to the nucleic acids to beprocessed.
 7. The method of claim 1, in which at least one processingstep requires a temperature that differs from the temperature of thereaction mixture.
 8. The method of claim 7, wherein atemperature-changing step serves for thermal denaturation.
 9. The methodof claim 7, wherein a temperature-changing step serves to hybridizenucleic acids.
 10. The method of claim 7, wherein a temperature-changingstep serves to extend a template molecule.
 11. The method of claim 7,wherein various processing steps serve to target sequence-dependentlyamplify a template molecule.
 12. The method of claim 11, wherein a nontemperature-resistant DNA polymerase is used for the amplification. 13.The method of claim 11, wherein a temperature-resistant DNA polymeraseis used for the amplification.
 14. The method of claim 11, whereinRNA-dependent RNA polymerase is used for the amplification.
 15. Themethod of claim 11, wherein RNA-dependent DNA polymerase is used for theamplification.
 16. The method of claim 11, wherein DNA ligase is usedfor the amplification.
 17. The method of claim 1, wherein the volume ofthe reaction mixture is between 20 μl and 1 ml.
 18. A system forprocessing nucleic acids in a reaction mixture, comprisinga reactionmixture containing nucleic acids to be detected and reactants forprocessing the nucleic acids in a processing procedure, and atemperature-changing element which effects temperature changes in atemperature-changing zone of the reaction mixture while maintaining theremaining portion of the reaction mixture at essentially isothermalconditions, wherein the temperature-changing element has a surface whichadjoins the temperature-changing zone, the temperature-changing zone isrelatively small as compared to the entire reaction mixture andcomprises an area less than 0.2 mm in length from the surface, andbinding means for binding agents for processing the nucleic acids arebound to the surface.
 19. The system of claim 18, wherein the bindingmeans is a chemical or physical binding reagent.
 20. The system of claim18, wherein the binding means is a nucleic acid binding reagent and theagents for processing the nucleic acids are nucleic acids that arecomplementary to at least a part of the nucleic acids to be processed.21. The system of claim 18, wherein the temperature-changing element istime-coordinated to effect time-coordinated temperature changes in thetemperature-changing zone.
 22. The system of claim 18, wherein thebinding of the agents is achieved by chemical or biospecific binding.23. The system of claim 18, wherein the binding means is magneticparticles.
 24. The system of claim 23, further comprising a magnet,located behind the surface, which induces a magnetic field to attractthe magnetic particles.
 25. The system of claim 18, wherein thetemperature-changing element is a single-use device.
 26. The system ofclaim 18, wherein only the surface of the temperature-changing elementis single-use, and the remainder of the temperature-changing element canbe used several times.
 27. The system of claim 18, wherein the surfaceprotrudes into the reaction mixture.
 28. The system of claim 27, whereinthe surface can be cooled and/or heated.
 29. The system of claim 18,wherein the temperature of the surface is regulated by a control unit.30. The system of claim 18, wherein the temperature-changing elementcomprises a heating element with a small heat capacity and a coolingelement with a high heat capacity.
 31. The system of claim 30, whereinthe heating element is heated ohmically.
 32. The system of claim 30,wherein the heating element is heated by rays.
 33. The system of claim32, wherein the rays are infrared rays.
 34. The system of claim 32,wherein the rays are laser-generated rays.
 35. The system of claim 18,wherein the surface is composed of a foil made of a heat-conductingmaterial.
 36. The system of claim 35, wherein the heat-conductingmaterial is metal.
 37. The system of claim 36, wherein theheat-conducting material is gold.